This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. With the rapidly expanding growth of applications of PDS in biomedical study, the field is encountering the roadblock of limited throughput. Often many (of the order of a hundred) samples have to be screened and studied under a wide range of experimental conditions in order to obtain the desired structure and details of the functional mechanisms. We have developed at ACERT a very sensitive and reliable pulse spectrometer operating at Ku band, which for a decade was arguably the most sensitive spectrometer anywhere for PDS. Also it has the unique capability of performing both of the main PDS pulse sequences: DEER (double electron-electron resonance) and DQC. At ACERT we have been addressing the issue of how to increase the throughput for PDS significantly under P41 ACERT Core Project I: "Protein Structure Determination by Pulse ESR". NIH supplementary funding is to enable us to achieve this important goal within the next two years. Our plans, which were outlined in our original proposal, are to increase throughput by a factor of 25 to 100 over what we currently achieve at ACERT on our high sensitivity Ku-band pulse ESR spectrometer, so that sufficient samples (up to about 100) could be studied in a single day of data acquisition, and this would typically be sufficient to complete a particular structure/function study. Thus, for example, an increase in sensitivity by one order of magnitude would reduce the data collection time by a factor of 100, i.e. it would be just a matter of a day to build a full triangulation network, upon which to determine the protein structure. This often suffices to resolve elements of secondary structure, to determine tertiary structure, and/or dock proteins into protein complexes. This would meet the criterion of high throughput (HT) technology for solving structures, with the capability of solving tens (or maybe hundreds) of proteins per year, limited only by the ability to produce large number of mutants. The basic approach that we plan to develop is to have parallel processing of a large number of samples with high sensitivity. The plan to develop HT PDS encompasses several specific aims, which are detailed in the subsequent seven time-ordered (but necessarily intertwined) individual subprojects (0276-0282). These aims are: (1) develop a system for automated data acquisition of consecutive samples at very low temperatures using batch processing;(2) increase the sensitivity of the pulse spectrometer by increasing the working frequency and optimizing the probe-head design;(3) implement a cryogenic receiver operating at a low temperatures to provide a very low noise temperature to further increase the sensitivity, thereby shortening data acquisition time;(4) construct a cryogen-free system that will operate continuously at temperatures in the range of 10-70 K;(5) extend the automated data acquisition to handle several samples simultaneously, thereby significantly increasing throughput;(6) improve the efficiency of sample handling and processing;(7)improve PDS data processing efficiency.